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Lu Lab - Protein Design

Intro block We design proteins.

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Introduction to the laboratory

       蛋白质设计(Protein Design)是合成生物学的重要分支和新兴的前沿学科,需要生物物理学、生物化学以及计算生物学等多学科的交叉融合。蛋白质设计是基于生物物理与生物化学原理,通过计算机模拟辅助,设计蛋白质氨基酸序列使其能够自发折叠形成设计预期的三维结构。人工设计的、具有全新结构和功能的蛋白质,将会广泛应用于生物医药和生物技术领域并产生深远影响。

       跨膜蛋白质的三维结构精确设计具有很大挑战性。卢培龙博士首次实现了多次跨膜蛋白三维结构的精确设计,并证明了计算机设计的蛋白质序列可以在膜环境中自发折叠形成与设计模型一致的结构。这一研究为设计具有全新结构和全新功能的跨膜蛋白铺平了道路,并可能对疫苗设计、DNA纳米孔测序、人工细胞信号环路设计等领域产生重要影响。

       实验室将自2019年开始招收博士研究生,现有多个博士后、助理研究员和科研助理职位虚位以待。期待共同为膜蛋白质设计这一新兴领域的发展作出贡献!

       西湖大学链接:

       西湖大学:社会力量举办、国家重点支持的新型高等学校

       新型高校激活一池春水---今后的路道阻且长,但我们坚信,未来,我们终将不辱使命!

       卢培龙实验室

       卢培龙博士入选《麻省理工科技评论》“35岁以下35人”中国榜单

 

        卢培龙研究组将致力于膜蛋白质相关设计这一多学科交叉领域的研究。

        实验室主要研究内容包括:

        (1)功能性膜蛋白质设计:主要包括新型跨膜纳米孔蛋白质、新型离子通道、以及新型膜受体的人工设计。

        (2)重大疾病相关膜蛋白质的拮抗蛋白质设计:拮抗蛋白质主要靶向作用于离子通道蛋白以及膜受体蛋白特定区域,并调控其生理功能。

        (3)为解决重大生物学问题设计新型蛋白质工具。

        这些研究将会极大地推动蛋白质设计领域的发展,为人类提供前所未有的新型方法和工具应用于生物医学研究,并在生物技术和生物医药领域有着广阔的科研转化前景,符合“中国制造2025”战略性新型产业发展的重大需求。

 

一、招聘岗位
岗位1:博士后/助理研究员
岗位职责:
围绕实验室研究方向开展科研工作, 并帮助和指导博士研究生。
应聘条件:
1.在相关领域近三年内获得或即将获得博士学位;
2.工作勤奋,细心负责,热爱科研,能独立地开展科研工作,具有良好的英语表达和论文写作能力,以第一作者身份发表过高水平SCI论文;
3.具备良好的沟通能力和团队协作精神。
  
岗位2:科研助理
岗位职责:
围绕实验室研究方向开展科研工作,并协助实验室其他成员开展科研工作。
应聘条件:
1.具有硕士及以上学历;
2.勤奋认真、积极主动,思路清晰、表达能力强,有服务意识、大局意识和团队意识;
3.有实验室相关工作经验者优先考虑。

二、 酬薪待遇
试用期考察通过后,将按工作能力和西湖大学的有关规定从优发放,享受五险一金及其他西湖大学和课题组提供的福利。在西湖大学的大力支持下,课题组将提供稳定的工作环境与一流的研究平台,会根据兴趣与需求支持个人的职业发展。
 
三、 应聘方法
1. 提供详细的个人简历(教育背景、工作经历、发表文章等)、毕业证明以及其他申请人认为必要的材料;
2. 提供2位推荐人的基本信息及联系方式;
3. 请将以上材料合并为一个PDF文件,发送至lupeilong@westlake.edu.cn,邮件标题请注明:应聘岗位+本人姓名+学历学位。
对于符合要求并通过初审者,将会通知安排面试。招聘启事在岗位招满前有效。

Nature发文报道丨世界上首次实现跨膜孔蛋白的精确从头设计

北京时间8月26日23时,Nature杂志在线发表西湖大学生命科学学院卢培龙研究员课题组与华盛顿大学David Baker等课题组合作的人工设计跨膜蛋白质的最新研究:《跨膜孔蛋白的计算机辅助设计》(Computational Design of Transmembrane Pores)该研究在世界上首次实现了跨膜孔蛋白的精确从头设计。

 

华盛顿大学徐纯福博士和西湖大学生命科学学院卢培龙研究员为该文的共同第一作者, 卢培龙研究员、华盛顿大学William A. Catterall教授和David Baker教授为该文的共同通讯作者。此外,大阪大学、剑桥大学的多位研究人员也在该项研究中作出重要贡献。

 

Part 1

从三个基本概念说起

 

要理解这项研究,首先要从3个基本概念说起:膜蛋白、通道蛋白/跨膜孔蛋白和蛋白质设计。

 

膜蛋白是指生物膜上的蛋白质,是生物膜功能的主要承担者,介导了细胞与外界环境之间的物质交换与信息传递,并且是能量代谢的重要参与者。如果我们把细胞想象成一间屋子,那膜蛋白就是这间屋子的窗户,阳光、空气不断通过这个屋子的不同窗户与室内进行交换。

 

通道蛋白,是膜蛋白的一种,相当于这间屋子其中一扇窗户,作为物质跨膜转运的通道,它在神经信号传递、细胞程序性死亡等复杂的生理活动中起到了至关重要的作用,是很多重大人类疾病的药物作用靶点,也作为蛋白质工具被广泛应用于生物技术与研究。本次研究所设计的跨膜孔蛋白,就隶属于通道蛋白。

 

蛋白质设计是合成生物学领域的核心技术和新兴的前沿学科。蛋白质设计通过编排蛋白质的氨基酸序列,使其能够自发折叠形成所需要的三维结构,并具有一定的功能。蛋白质的从头设计,即完全基于生物物理与生物化学原理,不依赖现有的天然蛋白质结构,从头搭建、设计具有全新结构和全新功能的蛋白质,可以帮助我们探索整个蛋白质序列折叠空间。较之大自然界演化的蛋白质,人工设计的蛋白质,能在性能方面更好满足我们特定的需求。

 

Part 2

卢培龙他们做了什么?

 

在本项研究中,卢培龙实验室与合作团队一起,成功设计了由两层ɑ螺旋同心环组成的2种跨膜孔蛋白(图1),分别可以选择性通透不同分子尺寸以及带电性质的溶质。

 

图1. 两层ɑ螺旋同心环组成的跨膜孔蛋白结构示意图

 

首先,研究人员通过对ɑ螺旋结构进行参数化设计,设计了由12个螺旋和16个螺旋组成的水溶性形式的孔蛋白。其中,12螺旋的孔蛋白(六聚体)孔径约为3.3 Å ,16螺旋的孔蛋白(八聚体)孔径约为10 Å。通过对设计孔蛋白进行重组表达、纯化、鉴定与结构验证, 研究人员证明所设计的孔蛋白性质非常稳定(比如结构较之天然蛋白,具备对高温更好的耐受性),并具有与计算设计模型相一致的三维结构。

 

 图2.本研究所设计的16个螺旋组成的跨膜孔蛋白的3D打印模型

 

在此基础上,研究人员设计了相应的跨膜孔蛋白。电生理实验表明,12螺旋跨膜通道蛋白可以通透离子,并且具有对钾离子的选择性;换句话说,这种蛋白可以特异性选择通透某一种离子。在脂质体实验中,16螺旋跨膜纳米孔蛋白可以通透分子量约为1000道尔顿的荧光分子,而12螺旋通道蛋白则不能;即该种孔蛋白作为“筛子”,也对分子的空间大小有所要求,当分子满足这个“孔”的“大小”,就可以穿透;这也与两种孔蛋白各自的孔径相符。最后,研究人员解析了16螺旋跨膜纳米孔蛋白的冷冻电镜结构,与设计模型非常一致,证明了所开发的从头设计方法的准确性。

 

这项研究是世界上第一次实现对跨膜孔蛋白质的精确从头设计,有助于人们更好地理解物质跨膜转运,即细胞在新陈代谢等生命活动过程中进行正常物质交换的基本原理,为人工设计具有重要功能的跨膜蛋白质奠定了坚实的基础。

 

这也为人工蛋白质后续可能的应用打开了大门,有望为纳米孔基因测序、分子检测等生物技术提供新的检测手段。例如:人工设计具有特殊通道结构的纳米孔蛋白,可应用于纳米孔测序技术,提高DNA纳米孔测序技术的精度;人工设计全新配体门控的通道蛋白,将能推进基于通道蛋白的分子检测技术等。相当于我们可以在一间屋子里设计不同的“窗户”,实现不同的功能。

 

 

Part 3

最大的挑战是“控制它的形状”

 

卢培龙长期致力于蛋白质设计方向的研究,早在2018年,他就实现了多次跨膜蛋白三维结构的精确设计,证明了计算机设计的蛋白质序列可以在膜环境中自发折叠形成与设计模型一致的稳定三维结构(研究成果发表于Science杂志https://science.sciencemag.org/content/359/6379/1042)。本次研究是基于以往研究成果取得的最新突破。

 

图3.卢培龙研究员与跨膜孔蛋白模型

 

它的难度在于,跨膜孔蛋白/通道蛋白与跨膜蛋白一样,都属于膜蛋白,但是具有更大的比表面积和相对低密度的分子内相互作用,从头设计跨膜孔蛋白的难度更为艰巨;同时,进一步来讲,如何在设计跨膜孔蛋白结构基础之上,实现选择性离子转运和小分子通透的功能,也面临着巨大挑战。也就是说,在研究过程中,如何设计氨基酸的序列排布,自发形成特定结构的孔蛋白,让在微小尺寸上的蛋白质“长成特定的样子”,并具有特定的转运功能,是研究的重难点。

 

研究团队进行过两个版本的设计,首个版本的孔蛋白在空间上具有更为卷曲的超螺旋,因此并没有形成稳定的孔蛋白;他们对设计进行了持续的改进、验证,对孔蛋白空间结构参数进行了调整,最终获得了成功。

 

接下来,西湖大学卢培龙研究组将继续通过蛋白质设计,为人类提供全新的蛋白质设计方法和自然界中不存在的蛋白质工具,满足生物技术与生物医学领域的需求。

 

在这项研究中,蛋白质从头设计工作得到西湖大学高性能计算平台的支持,冷冻电镜数据采集于西湖大学冷冻电镜平台,蛋白质质谱分析完成于西湖大学质谱平台;本工作获得了国家自然科学基金委、西湖实验室、腾讯基金会的经费支持。

 

全文链接:

https://www.nature.com/articles/s41586-020-2646-5

卢培龙博士入选《麻省理工科技评论》“35岁以下35人”中国榜单

卢培龙

年龄:31 岁

职位:西湖大学生命科学学院PI

获奖理由:他首次实现了多次跨膜蛋白三维结构的精确设计。

 

蛋白质设计是结构生物学的重要分支和新兴的前沿学科,需要生物物理学、生物化学、合成生物学以及计算生物学等多学科的交叉融合。

卢培龙通过计算生物学手段模拟蛋白质极性残基在膜环境内部形成的相互作用,设计了能够在膜环境中稳定存在的蛋白质三维结构,并进行重组表达、生化性质测定、三维结构解析等一系列实验来进行验证。

卢培龙利用这一方法成功设计了多种具有极高热稳定性的跨膜蛋白质,并证明了计算机设计的蛋白质序列可以在膜环境中自发折叠形成与设计模型一致的结构。

该研究在世界上首次实现了多次跨膜蛋白三维结构的精确设计,为设计具有全新结构和全新功能的跨膜蛋白铺平了道路,并可能对疫苗设计、DNA 纳米孔测序、人工细胞信号环路设计等重要领域产生深远影响。

Keep Your Eyes on the Prize: A Life Connected with Science -------- By Ronald D. Vale

"I feel that my greatest prize has been the privilege of a life connected with science. I have enjoyed innumerable discoveries, both my own and those of others, and I have met many kind and interesting people along the way. My best advice to young scientists is to keep your eyes on these prizes. If you do, everything else will fall into place."  --------- Ronald D. Vale

https://www.cell.com/cell/fulltext/S0092-8674(19)30340-X

It is a great honor for me to receive the prestigious Gairdner Award for Biomedical Research. The title of this essay might imply that it will contain advice on how to win such a major scientific prize. However, my intention is quite different. More in keeping with the famous civil rights song of the same name, my hope is to articulate a bigger picture view of what matters in the long run. Why do we do science? What is fulfilling about a career in science? Is an international prize the pinnacle of success, or are there more important outcomes?

Prizes recognize fortunate scientists who have unearthed some amazing attribute of living organisms. Under slightly different circumstances and perhaps a bit later, another scientist would have uncovered the same treasure. Thus, winning a major prize is unpredictable; there are too many contingent factors beyond one’s control. Most readers of this article will not win a major prize. However, most scientists will experience the joy of discovery. Unlike awards, which are few in number, discoveries are not uncommon. Unlike international awards, discoveries are bestowed upon undergraduates as well as senior scientists. Even small discoveries can be immensely rewarding. A discovery, along with the people with whom it can be shared, is a “prize” worthy of our attention and aspiration. Such satisfaction is accessible to many people in the scientific profession, if they are cognizant about keeping their eyes on the prize.

 Enjoying Discovery

During high school, many students perform laboratory experiments that are intended mostly to reproduce known outcomes (and one’s grade might suffer if results deviate from the expected). I remember the first time I did a real experiment for a science fair project for my biology class at Hollywood High School. I read somewhere that plants and animals could tell time and that these circadian rhythms govern many of their behaviors. How amazing! I first wanted to measure a circadian rhythm myself, specifically the up-and-down motions of the leaves of a bean plant during day and night (see movie). Clearing out a space in our cluttered basement, I built a kymograph consisting of a motor that slowly rotated a coffee can with paper wrapping its exterior; an ink pen, attached via a string to the plant leaf, would trace the motion of the leaf on the rotating paper. It was not very complicated, but I was terrifically pleased that I got it to work.

With this initial success under my belt, I then thought it would be fun to trick the clock and see how it responded to light-dark cycles that strayed from the 24 hour day. What would happen if I turned the lights on and off in my basement every 6 hours? It was the first time I tried to pose my own question and answer it. That was the start of my scientific career. It was a thrill to be on the hunt for knowledge, rather than just memorizing it. I have had a career of posing curiosity-driven questions ever since.

When it came time to present what I thought was an awesome biology project, my biology teacher did not award me with the top grade (similar to “referee 3” of some of my latter scientific papers). However, I expressed my enthusiasm for the project to my high school counselor Ella Hogan. Ella took the initiative of calling a laboratory at UCLA working on circadian rhythms to see if they would take in a high school student who was keen on doing research. The professor (Dr. Karl Hammer) agreed to let me work in his laboratory, and I graduated from my basement to doing experiments in a real laboratory in the Department of Plant Physiology. At the start, my mother patiently drove me—before I had my independent driver's license— to UCLA. Being in high school in those days (1970s) was different than today. Students were not seeking internships and trying to amass multi-page CVs to look good for colleges. I was just doing research because it was interesting and fun (along with other things that teenage kids did in Hollywood, California, at the time).

The point of this tale is that I enjoyed measuring bean plant movements. In fact, from my memory, I enjoyed doing science as much in high school as I do now. Naturally, the level of science has advanced. The home-built kymograph has been supplanted by lab-built single-molecule and light sheet microscopes. My presentations have advanced from science fairs to international conferences. Rudimentary high school results evolved into findings recognized by the Gairdner Award jury. However, I always enjoyed what I was capable of doing at the time. You don’t need to be a Gairdner Award winner to enjoy science.

 Cherish Small Victories

Setting one’s sights on answering an interesting scientific question is important. However, nature does not give up its secrets readily. As students advance from college to graduate school, one of the hardest lessons they learn is that many experiments don’t work and that science takes time. The rapid and (for achievers) positive feedback of mid-terms and finals is replaced by the real world situation of conquering the unknown. When it comes to obtaining interpretable results, nature does not grade on a bell curve. Students have to learn patience and resilience, which is as important as learning new techniques. Fortunately, my strongest attributes are patience and determination, which have served me well in my scientific career.

Where is the pleasure in an endeavor that is marked by more failures than victories; where the final goal is a scientific paper that often takes years to produce; where jobs and career advancement are not guaranteed? The answer is in small victories—they are all around if you look for them. The protein preparation that finally worked; arriving at the right salt concentration for your experiment; having the microscope stay in focus for the entire time-lapse movie; etc. Not big results perhaps, but they are personal victories. A moment to congratulate oneself; a good day in the lab to savor. These small victories are the foundation of science; collectively they add up to a successful scientific career. You cannot bypass them on the way to a big result. Importantly, you don’t need to wait for years until the acceptance of a paper or decades until a Gairdner award. Fortunately, pleasure and satisfaction in a scientific project can happen more frequently and to many more people, and one should aim to recognize and acknowledge such moments when they happen.

 People Are More Important than Papers

Science is all about the data. Or is it? In addition to results, science is a rich network of social interactions. A well-functioning scientific laboratory is like a family. It is a place of wonderful social interactions and an environment that provides a support structure. Beyond the laboratory, scientists establish deep friendships across the globe, bonds created by shared interests in science as well as by compatible personalities. These interactions have lasting and important value. Friendships established in a laboratory are often friendships for life. Collaborators frequently continue their friendship well beyond an initial connection made through a mutual need of obtaining results. Young scientists remember their kind mentors for their entire lives. Senior scientists are gratified when their mentees return to share their appreciation and memories.

So much attention now is focused on obtaining “high impact” papers, unfortunately measured by publication in a very limited set of journals. A great paper can make a splash and influence the work of other scientists (an important mark of success). However, that high profile paper might be largely forgotten after a few years. Friendships in science, however, can easily last years and often a lifetime. Scientists who understand this and have nurtured these interactions have received a valuable “prize”. They are well-connected and valued by other individuals in the scientific community. This prize is accessible to many scientists, junior and senior, provided that they are mindful of the value of people and not just papers.

 Paying It Forward

If we, as scientists, manage to climb up one rung on the career ladder, it is our responsibility to reach down with the other hand and pull the next person up. In addition to being a responsibility, “paying it forward” also creates a sense of well-being and contribution.

I shared a story about my high school counselor; her time and attention had an impact, as it shaped my life and career. I had little to offer her concretely at the time. However, I think that my keen interest and appreciation was rewarding to her. Now that I am older, my career has shifted from being a recipient of opportunity to one who now can focus on supporting others. I make few discoveries with my own hands these days (my last significant one being in 2013). My own hunt for discovery has evolved into vicarious enjoyment of watching trainees go through that process. That role has its own gratification. Seeing young scientists become engaged with science—that is fun! Having a postdoctoral fellow move on to their dream job—that is satisfying!

You don’t need to be famous, be at prestigious university, or win the Gairdner Award to pay it forward. You don’t even need to wait to reach my ripe old age. College students can inspire and mentor high school students. Graduate students can help college students. And so on. There is always some one who will benefit from your attention. This prize in science is awarded to all who show kindness and are willing to look beyond their own career to help others.

 The Opportunity to Keep Learning by Enjoying Other People’s Discoveries

There is only so much that one can discover oneself. A year may go by without an exciting result. Fortunately, the overall scientific enterprise is creating a continuous stream of exciting results. Why not enjoy them too?

I enjoy being a scientist, because of the extraordinary opportunity to keep learning. I am a student at heart, and the scientific profession embodies this philosophy in the duties of the job. I am continually amazed by the inner workings of living organisms and their seemingly limitless adaptations. These interesting features of life are being unraveled at a dizzying pace. I feel fortunate to have been trained with the appropriate knowledge and skills to allow me to appreciate the life sciences in these exciting times. It is a joy to listen to a fantastic seminar or read about a clever technique developed by another group. I am happy to hear about science being done beyond the boundaries of my own laboratory. This prize is accessible to undergraduates and Gairdner Award winners alike. We all can enjoy hearing about new results and be in awe of the wondrous attributes of life.

 Conclusion

I am very fortunate to have received the Gairdner Award in Biomedical Research, which recognizes approximately two decades of work on the motor protein kinesin from its discovery to the dissection of its mechanism of movement. Without diminishing the honor that I feel on having been selected for this award, I feel that my greatest prize has been the privilege of a life connected with science. I have enjoyed innumerable discoveries, both my own and those of others, and I have met many kind and interesting people along the way. My best advice to young scientists is to keep your eyes on these prizes. If you do, everything else will fall into place.

 

De novo Protein Design---Science's Breakthrough of the Year, 2016

 Cover image expansion

"Designing new proteins from scratch has been a hit-or-miss activity. It’s easy enough to write any desired DNA code, but researchers have had no way of knowing how the novel strings of amino acids encoded by this DNA would fold into complex 3D shapes. That’s a problem, because for proteins, shape dictates function. Recently, however, computational biologists have made heady progress in designing computer programs that accurately predict how designer proteins will fold. Those advances made possible this year’s surge in designer proteins."

http://www.sciencemag.org/news/2016/07/protein-designer-aims-revolutionize-medicines-and-materials

 

Accurate computational design of multipass transmembrane proteins.

The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.

http://science.sciencemag.org/content/359/6379/1042.long

 

Computational design of transmembrane pores

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2–4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7 ,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore—but not the 12-helix pore—enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

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